The present invention relates to a method for the mutual separation of proteins, i.e. the separation of proteins from each other.
It is well known that various physioactive proteins, such as cytokines and peptide hormones, exist. The current progress of gene engineering techniques is making way for the mass production and clinical application of these physioactive proteins.
Such techniques have already been applied to provide production of a wide variety of physiologically active proteins, including cytokines, such as interferons, interleukins, B cell growth factor, B cell differentiation factor, macrophage activating factor, lymphotoxin, tumor necrosis factor, etc., peptide hormones, such as erythropoietines, epithelial cell growth factor, insulin, human growth hormone, transforming growth factor, etc., antigenic proteinaceous materials useful in developing vaccines against pathogenic microorganisms, such as hepatitus B virus antigen, influenza antigen, foot-and-mouth disease virus antigen, malarial parasite antigen, etc., enzymes such as peptidases (e.g., tissue plasminogen activator, urokinase, serratiopeptidase, etc.), lysozyme, etc., hemoproteins, such as human serum albumin, etc., and other proteins having useful physiological properties.
However, where such proteins have been produced by recombinant methods, there have been problems experienced in separating the ultimate desired product from other materials with which it is often mixed. A particularly vexing problem has been caused by the fact that the expression product obtained by expression of the gene coding for the protein of interest often contains a mixture of the protein of interest and a second protein, the second protein comprising the protein of interest bound to an extraneous methionine group at its amino terminus The extraneous methionine (Met) group comes from the expression of the ATG start codon which signals the start of expression of the desired gene, and the failure of the expression system of the host cell to excise the extraneous Met group from the expression product. This problem can occur in both prokaryotic hosts and eukaryotic hosts, but most often occurs in expression of genes in prokaryotic hosts. It is a particular problem in expression systems utilizing Eschericia coli as expression host.
Because protein synthesis in both eukaryotes and prokaryotes starts at the mRNA codon AUG, which codes for the amino acid methionine, it is not unexpected that the protein expression produces a mixture of molecular species, namely the protein of interest and its analogs with an extraneous Met at the N-terminus (N-Met analog), particularly when the expression host is Escherichia coli.
In fact, it is known that IF-3, an Escherichia coli initiation factor, includes two molecular species, i.e. the one species possessing a methionine residue at the amino terminus and the other without a mechionine as the terminal residue [Hoppe Seyler's Z. Physiol. Chem., 354, 1415 (1973)], and that the amino terminus of Escherichia coli proteins is commonly methionine [Conn & Stumpf (1976), Outlines of Biochemistry, 4th edition, John Wiley & Sons]. The problem of production of high ratios of the Met analog to the desired protein produced by recombinant DNA technology has also been widely experienced. See, e.g., Nature, 293, 408 (1981), wherein the production of human growth hormone resulted in a large ratio of the Met analog to the actual desired protein.
Where a mixture of the desired protein and its N-Met analog are produced, it is extremely difficult to separate one from the other, because the two molecular species differ little, if at all, in their physicochemical properties.
The methionine residue is an amino acid residue with a molecular weight of approximately 131 and with a medium hydrophobicity and it is electrically neutral because of the lack of dissociative groups. In addition, protein is a macromolecule possessing many dissociative groups, hydrophobic groups, and hydrophilic groups. For example, the molecular weight of the interleukin-2 polypeptide (I) shown in FIG. 1, which is composed of 133 amino acid residues (where X means a hydrogen atom), is approximately 15,420. Therefore, it is expected that the addition of one methionine residue to the amino terminus of protein normally does not greatly affect the physicochemical properties of the protein itself. Thus it can be extremely difficult to separate the molecular species to which a methionine residue has been added at the amino terminus and that without the terminal methionine residue from each other.
While this difficulty is common to many proteins, the difficulties have been particularly acute in attempts at separating recombinant interleukin-2 and recombinant interferon from their N-Met analogs, especially when the recombinant proteins have been expressed using Escherichia coli as the host for the expression.
Interleukin-2, one of the lymphokines which are produced by the T cells activated by mitogens or antigens, is a factor essential to the growth and differentiation of cytotoxic T cells and natural killer cells and it exerts an important action in the immunoreaction system mediated by these cells.
Interferon-.alpha., a kind of the lymphokines which are produced by the leukocytes activated by viruses or nucleic acids, has a bioactivity of acting on cells to put them into the antivirus state and it exerts an important action in the antiinfection system and oncoimmunity system.
It is expected that interleukin-2 and interferon-.alpha. can be effectively used as therapeutics for immune deficiency diseases, infectious diseases, malignant tumors etc. because of their bioactivities. The natural interleukin-2 which has been isolated from the supernatant of the culture of human peripheral blood lymphocytes or human T cell leukemia cells (Jurkat line), includes a few molecular species with different molecular weights; however, it is known that all of them resemble each other very much with regard to the polypeptide chain composition, e.g., their amino terminus is an alanine residue without exception [Japanese Patent Application No. 149248/1984 (Filing Date: Jul. 19, 1984) which corresponds to EP Publication (laid open) No. 032359; Pro. Natl. Acad. Sci. USA, 81, 2543 (1984)].
The natural interferon-.alpha. which has been isolated from the supernatant of the culture of human leukocytes, includes ten or more subtypes; however, all of them resemble to each other very much with regard to the polypeptide chain composition, e.g., their amino terminus is a cysteine residue without exception [Arch. Biochem. Biophys., 221, 1, (1983)].
The present inventors have succeeded in producing non-glycosylated human interleukin-2 by expressing the human lymphocyte interleukin-2 gene in Escherichia coli cells by means of recombinant DNA technology [Japanese Patent Application No. 225079/1983 (Filing Date: Nov. 28, 1983) which corresponds to EP Publication (laid open) No. 0145390]. The said interleukin-2 contains polypeptide (I), which possesses the amino acid sequence shown in FIG. 1 (in the figure, X means a hydrogen atom or a methionine residue), and it includes two molecular species, i.e., the one species whose amino terminus is an alanine residue as in natural human interleukin-2 (i.e. X is a hydrogen atom) and the other whose amino terminus is a methionylalanine residue to which a methionine residue has been added at the amino terminus (i.e. X is a methionine residue).
As previously reported [J. Interferon Res., 1, 381 (1981); J. Biol. Chem., 256, 9750 (1981)], interferon-.alpha.A which has been expressed in Escherichia coli cells by means of recombinant DNA technology, for example, contains the polypeptide possessing the amino acid sequence shown in FIG. 2, and it includes two molecular species, i.e. the one species whose amino terminal amino acid is a cysteine residue as in natural human interferon-.alpha. (i.e. X is a hydrogen atom) and the other whose amino terminal amino acid is a methionyl-cysteine residue to which a methionine residue has been added at the amino terminus (i.e. X is a methionine residue).
It is possible that the highly ordered conformation of protein differs between the molecular species to which a methionine residue has been added at the amino terminus and that without a methionine as the terminal residue; therefore, it is also possible that the bioactivities or biological stability in vivo and in vitro are different between the two. In addition, the addition of a methionine residue to the amino terminus may cause the increase or decrease in antigenicity. Consequently, from the point of view of physiological and industrial application, it is highly desirable to obtain practically pure molecular species by separating the molecular species to which a methionine residue has been added at the amino terminus and that without a methionine as the terminal residue from each other.
The ratio of the addition of a methionine residue to the amino termini may be affected by culture conditions or protein expression levels [J. Interferon Res., 1, 381 (1981)]; however, no example has been reported yet where the ratio of the addition of a methionine residue was successfully controlled. Likewise, no example has been reported yet where the molecular species to which a methionine residue had been added at the amino terminus and that without the terminal methionine residue were separated from each other in the protein purification process.
The present inventors could not separate either (a) an interleukin-2 possessing a methionine residue at the amino terminus and the protein without the terminal methionine residue, or (b) the interferon-.alpha.A possessing a methionine residue at the amino terminus and that without the methionine terminal residue, from each other, respectively, although many methods were tried as a method for the mutual separation, e.g. salting-out and solvent precipitation, where the difference of solubility is utilized, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, where the difference of molecular weight is mainly utilized, affinity chromatography which utilizes the specific affinity of antibody for the proteins, and reversed-phase high performance liquid chromatography, where the difference of hydrophobicity is utilized.